Complementary Fragmentation Techniques Research Articles

Enrichment of N-Linked Glycopeptides and Their Identification by Complementary Fragmentation Techniques.

N-linked glycans are a ubiquitous posttranslational modification and are essential for correct protein folding in the endoplasmic reticulum of plants. However, this likely represents a narrow functional role for the diverse arrayof glycan structures currently associated with N-glycoproteins in plants. The identification of N-linked glycosylation sites and their structural characterization by mass spectrometry remains challenging due to their size, relative abundance, structural heterogeneity, and polarity. Current proteomic workflows are not optimized for the enrichment, identification and characterization of N-glycopeptides. Here we describe a detailed analytical procedure employing hydrophilic interaction chromatography enrichment, high-resolution tandem mass spectrometry employing complementary fragmentation techniques (higher-energy collisional dissociation and electron-transfer dissociation) and a data analytics workflow to produce an unbiased high confidence N-glycopeptide profile from plant samples.

Characterization of protein N-glycosylation by tandem mass spectrometry using complementary fragmentation techniques

The analysis of post-translational modifications (PTMs) by proteomics is regarded as a technically challenging undertaking. While in recent years approaches to examine and quantify protein phosphorylation have greatly improved, the analysis of many protein modifications, such as glycosylation, are still regarded as problematic. Limitations in the standard proteomics workflow, such as use of suboptimal peptide fragmentation methods, can significantly prevent the identification of glycopeptides. The current generation of tandem mass spectrometers has made available a variety of fragmentation options, many of which are becoming standard features on these instruments. We have used three common fragmentation techniques, namely CID, HCD, and ETD, to analyze a glycopeptide and highlight how an integrated fragmentation approach can be used to identify the modified residue and characterize the N-glycan on a peptide.

Glycoproteomic Analysis of Seven Major Allergenic Proteins Reveals Novel Post-translational Modifications

Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM(1) characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. We demonstrate the presence of lysine-linked polyhexose glycans and asparagine-linked N-acetylhexosamine glycans on HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted desensitization strategies and improved allergy vaccines.

Targeting the glycoproteome

Despite numerous original publications describing the structural complexity of N- and O-linked glycans on glycoproteins, only very few answer the basic question of which particular glycans are linked to which amino acid residues along the polypeptide chain. Such structural information is of fundamental importance for understanding the biological roles of complex glycosylations as well as deciphering their non-template driven biosynthesis. This review focuses on presenting and commenting on recent strategies, specifically aimed at identifying the glycoproteome of cultured cells and biological samples, using targeted and global enrichment procedures and utilizing the high resolution power, high through-put capacity and complementary fragmentation techniques of tandem mass spectrometry. The goal is to give an update of this emerging field of protein and glyco-sciences and suggest routes to bridge the data gap between the two aspects of glycoprotein characteristics, i.e. glycan structures and their attachment sites.

Phosphorylation, but Not Alternative Splicing or Proteolytic Degradation, Is Conserved in Human and Mouse Cardiac Troponin T

Cardiac troponin T (cTnT), the tropomyosin binding subunit of the troponin complex, plays a pivotal regulatory role in the Ca(2+)-mediated interaction between actin thin filament and myosin thick filament. The post-translational modifications (PTMs) and alternative splicing of cTnT may represent important regulatory mechanisms of cardiac contractility. However, a complete characterization of PTMs and alternatively spliced isoforms in cTnT present in vivo is lacking. Top-down protein mass spectrometry (MS) analyzes whole proteins, thus providing a global view of all types of modifications, including PTMs and sequence variants, simultaneously in one spectrum without a priori knowledge. In this study, we applied an integrated immunoaffinity chromatography and top-down MS approach to comprehensively characterize PTMs and alternatively spliced isoforms of cTnT purified from healthy human and wild-type mouse heart tissue. High-resolution Fourier transform MS revealed that human cTnT (hcTnT) and mouse cTnT (mcTnT) have similar phosphorylation patterns, whereas higher molecular heterogeneity was observed for mcTnT than hcTnT. Further MS/MS fragmentation of monophosphorylated hcTnT and mcTnT by electron capture dissociation and collisionally activated dissociation unambiguously identified Ser1 as the conserved in vivo phosphorylation site. In contrast, we identified a single spliced isoform for hcTnT but three alternatively spliced isoforms for mcTnT. Moreover, we observed distinct proteolytic degradation products for hcTnT and mcTnT. This study also demonstrates the advantage of top-down MS/MS with complementary fragmentation techniques for the identification of modification sites in the highly acidic N-terminal region of cTnT.

The Generating Function of CID, ETD, and CID/ETD Pairs of Tandem Mass Spectra: Applications to Database Search

Recent emergence of new mass spectrometry techniques (e.g. electron transfer dissociation, ETD) and improved availability of additional proteases (e.g. Lys-N) for protein digestion in high-throughput experiments raised the challenge of designing new algorithms for interpreting the resulting new types of tandem mass (MS/MS) spectra. Traditional MS/MS database search algorithms such as SEQUEST and Mascot were originally designed for collision induced dissociation (CID) of tryptic peptides and are largely based on expert knowledge about fragmentation of tryptic peptides (rather than machine learning techniques) to design CID-specific scoring functions. As a result, the performance of these algorithms is suboptimal for new mass spectrometry technologies or nontryptic peptides. We recently proposed the generating function approach (MS-GF) for CID spectra of tryptic peptides. In this study, we extend MS-GF to automatically derive scoring parameters from a set of annotated MS/MS spectra of any type (e.g. CID, ETD, etc.), and present a new database search tool MS-GFDB based on MS-GF. We show that MS-GFDB outperforms Mascot for ETD spectra or peptides digested with Lys-N. For example, in the case of ETD spectra, the number of tryptic and Lys-N peptides identified by MS-GFDB increased by a factor of 2.7 and 2.6 as compared with Mascot. Moreover, even following a decade of Mascot developments for analyzing CID spectra of tryptic peptides, MS-GFDB (that is not particularly tailored for CID spectra or tryptic peptides) resulted in 28% increase over Mascot in the number of peptide identifications. Finally, we propose a statistical framework for analyzing multiple spectra from the same precursor (e.g. CID/ETD spectral pairs) and assigning p values to peptide-spectrum-spectrum matches.

Proteome Analysis of Sorangium cellulosum Employing 2D-HPLC-MS/MS and Improved Database Searching Strategies for CID and ETD Fragment Spectra

Shotgun proteome analysis of the myxobacterial model strain for secondary metabolite biosynthesis Sorangium cellulosum was performed employing off-line two-dimensional high-pH reversed-phase HPLC x low-pH ion-pair reversed-phase HPLC and dual tandem mass spectrometry with collision-induced dissociation (CID) and electron transfer dissociation (ETD) as complementary fragmentation techniques. Peptide identification using database searching was optimized for ETD fragment spectra to obtain the maximum number of identifications at equivalent false discovery rates (1.0%) in the evaluation of both fragmentation techniques. In the database search of the CID MS/MS data, the mass tolerance was set to the well-established 0.3 Da window, whereas for ETD data, it was widened to 1.1 Da to account for hydrogen-rearrangement in the radical-intermediate of the peptide precursor ion. To achieve a false discovery rate comparable to the CID results, we increased the significance threshold for peptide identification to 0.001 for the ETD data. The ETD based analysis yielded about 74% of all peptides and about 78% of all proteins compared to the CID-method. In the combined data set, 952 proteins of S. cellulosum were confidently identified by at least two peptides per protein, facilitating the study of the function of regulatory proteins in the social myxobacteria and their role in secondary metabolism.

Two Dimensional Mass Mapping as a General Method of Data Representation in Comprehensive Analysis of Complex Molecular Mixtures

A recent proteomics-grade (95%+ sequence reliability) high-throughput de novo sequencing method utilizes the benefits of high resolution, high mass accuracy, and the use of two complementary fragmentation techniques collision-activated dissociation (CAD) and electron capture dissociation (ECD). With this high-fidelity sequencing approach, hundreds of peptides can be sequenced de novo in a single LC-MS/MS experiment. The high productivity of the new analysis technique has revealed a new bottleneck which occurs in data representation. Here we suggest a new method of data analysis and visualization that presents a comprehensive picture of the peptide content including relative abundances and grouping into families. The 2D mass mapping consists of putting the molecular masses onto a two-dimensional bubble plot, with the relative monoisotopic mass defect and isotopic shift being the axes and with the bubble area proportional to the peptide abundance. Peptides belonging to the same family form a compact group on such a plot, so that the family identity can in many cases be determined from the molecular mass alone. The performance of the method is demonstrated on the high-throughput analysis of skin secretion from three frogs, Rana ridibunda, Rana arvalis, and Rana temporaria. Two dimensional mass maps simplify the task of global comparison between the species and make obvious the similarities and differences in the peptide contents that are obscure in traditional data presentation methods. Even biological activity of the peptide can sometimes be inferred from its position on the plot. Two dimensional mass mapping is a general method applicable to any complex mixture, peptide and nonpeptide alike.

Combining bottom-up and top-down mass spectrometric strategies for de novo sequencing of the crustacean hyperglycemic hormone from Cancer borealis.

The crustacean hyperglycemic hormone (CHH) is a 72-amino acid residue polypeptide with multiple physiological effects. The X-organ/sinus gland is the primary source for CHH and its family members. However, the amino acid sequence of CHH in Cancer borealis , a premier model system for neuromodulation, has not been characterized. In this study, a novel hybrid strategy combining "bottom-up" and "top-down" methodologies enabled direct sequencing of CHH peptide in the sinus gland of C. borealis . Multiple mass spectrometry (MS)-based techniques were employed to characterize the CHH peptide, including direct tissue analysis by MALDI-FT-ICR-MS, de novo sequencing of tryptic digested CHH by nano-LC/ESI-Q-TOF MS and intact CHH analysis by LC/FT-ICR-MS. In-trap cleaning removed the extensive matrix adducts of CHH in the direct tissue analysis by MALDI-FT-ICR-MS. Fragmentation efficiency of the intact CHH was drastically improved after the reduction-alkylation of the disulfide bonds. The sequence coverage was further enhanced by employing multiple complementary fragmentation techniques. Overall, this example is the largest neuropeptide de novo sequenced in C. borealis by mass spectrometric methods.

Phosphorylation of SUMO-1 Occurs in Vivo and Is Conserved through Evolution

Protein dynamics is regulated by an elaborate interplay between different post-translational modifications. Ubiquitin and ubiquitin-like proteins (Ubls) are small proteins that are covalently conjugated to target proteins with important functional consequences. One such modifier is SUMO, which mainly modifies nuclear proteins. SUMO contains a unique N-terminal arm not present in ubiquitin and other Ubls, which functions in the formation of SUMO polymers. Here, we unambiguously show that serine 2 of the endogenous SUMO-1 N-terminal protrusion is phosphorylated in vivo using very high mass accuracy mass spectrometry at both the MS and the MS/MS level and complementary fragmentation techniques. Strikingly, we detected the same phosphorylation in yeast, Drosophila and human cells, suggesting an evolutionary conserved function for this modification. The nearly identical human SUMO-2 and SUMO-3 isoforms differ in serine 2; thus, only SUMO-3 could be phosphorylated at this position. Our finding that SUMO can be modified may point to an additional level of complexity through modifying a protein-modifier.

A Sulfated, Phosphorylated 7 kDa Secreted Peptide Characterized by Direct Analysis of Cell Culture Media

An unusual sulfotyrosine-, phosphoserine-containing motif was mapped on a differentially post-translationally modified 60 residue antimicrobial neuroendocrine peptide called chrombacin. The study was performed by high resolution FT MS using complementary fragmentation techniques. The peptide was analyzed at low levels directly from cell culture media in contrast to previous reports that required extensive purification and proteolytic digestion. The sulfation site was not previously described nor predicted by informatic analysis of the peptide's precursor sequence.

Extent of Modifications in Human Proteome Samples and Their Effect on Dynamic Range of Analysis in Shotgun Proteomics

The complexity of the human proteome, already enormous at the organism level, increases further in the course of the proteome analysis due to in vitro sample evolution. Most of in vitro alterations can also occur in vivo as post-translational modifications. These two types of modifications can only be distinguished a posteriori but not in the process of analysis, thus rendering necessary the analysis of every molecule in the sample. With the new software tool ModifiComb applied to MS/MS data, the extent of modifications was measured in tryptic mixtures representing the full proteome of human cells. The estimated level of 8-12 modified peptides per each unmodified tryptic peptide present at >or=1% level is approaching one modification per amino acid on average. This is a higher modification rate than was previously thought, posing an additional challenge to analytical techniques. The solution to the problem is seen in improving sample preparation routines, introducing dynamic range-adjusted thresholds for database searches, using more specific MS/MS analysis using high mass accuracy and complementary fragmentation techniques, and revealing peptide families with identification of additional proteins only by unfamiliar peptides. Extensive protein separation prior to analysis reduces the requirements on speed and dynamic range of a tandem mass spectrometer and can be a viable alternative to the shotgun approach.

PhosTShunter: A Fast and Reliable Tool to Detect Phosphorylated Peptides in Liquid Chromatography Fourier Transform Tandem Mass Spectrometry Data Sets

A database independent search algorithm for the detection of phosphopeptides is described. The program interrogates the tandem mass spectra of LC-MS/MS data sets regarding the presence of phosphorylation specific signatures. To achieve maximum informational content, the complementary fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD) are used independently for peptide fragmentation. Several criteria characteristic for peptides phosphorylated on either serine or threonine residues were evaluated. The final algorithm searches for product ions generated by either the neutral loss of phosphoric acid or the combined neutral loss of phosphoric acid and water. Various peptide mixtures were used to evaluate the program. False positive results were not observed because the program utilizes the parts-per-million mass accuracy of Fourier transform ion cyclotron resonance mass spectrometry. Additionally, false negative results were not generated owing to the high sensitivity of the chosen criteria. The limitations of database dependent data interpretation tools are discussed and the potential of the novel algorithm to overcome these limitations is illustrated.

ModifiComb, a New Proteomic Tool for Mapping Substoichiometric Post-translational Modifications, Finding Novel Types of Modifications, and Fingerprinting Complex Protein Mixtures

A major challenge in proteomics is to fully identify and characterize the post-translational modification (PTM) patterns present at any given time in cells, tissues, and organisms. Here we present a fast and reliable method ("ModifiComb") for mapping hundreds types of PTMs at a time, including novel and unexpected PTMs. The high mass accuracy of Fourier transform mass spectrometry provides in many cases unique elemental composition of the PTM through the difference DeltaM between the molecular masses of the modified and unmodified peptides, whereas the retention time difference DeltaRT between their elution in reversed-phase liquid chromatography provides an additional dimension for PTM identification. Abundant sequence information obtained with complementary fragmentation techniques using ion-neutral collisions and electron capture often locates the modification to a single residue. The (DeltaM, DeltaRT) maps are representative of the proteome and its overall modification state and may be used for database-independent organism identification, comparative proteomic studies, and biomarker discovery. Examples of newly found modifications include +12.000 Da (+C atom) incorporation into proline residues of peptides from proline-rich proteins found in human saliva. This modification is hypothesized to increase the known activity of the peptide.

Proteomics-Grade de Novo Sequencing Approach

The conventional approach in modern proteomics to identify proteins from limited information provided by molecular and fragment masses of their enzymatic degradation products carries an inherent risk of both false positive and false negative identifications. For reliable identification of even known proteins, complete de novo sequencing of their peptides is desired. The main problems of conventional sequencing based on tandem mass spectrometry are incomplete backbone fragmentation and the frequent overlap of fragment masses. In this work, the first proteomics-grade de novo approach is presented, where the above problems are alleviated by the use of complementary fragmentation techniques CAD and ECD. Implementation of a high-current, large-area dispenser cathode as a source of low-energy electrons provided efficient ECD of doubly charged peptides, the most abundant species (65-80%), in a typical trypsin-based proteomics experiment. A new linear de novo algorithm is developed combining efficiency and speed, processing on a conventional 3 GHz PC, 1000 MS/MS data sets in 60 s. More than 6% of all MS/MS data for doubly charged peptides yielded complete sequences, and another 13% gave nearly complete sequences with a maximum gap of two amino acid residues. These figures are comparable with the typical success rates (5-15%) of database identification. For peptides reliably found in the database (Mowse score > or = 34), the agreement with de novo-derived full sequences was >95%. Full sequences were derived in 67% of the cases when full sequence information was present in MS/MS spectra. Thus the new de novo sequencing approach reached the same level of efficiency and reliability as conventional database-identification strategies.

New Data Base-independent, Sequence Tag-based Scoring of Peptide MS/MS Data Validates Mowse Scores, Recovers Below Threshold Data, Singles Out Modified Peptides, and Assesses the Quality of MS/MS Techniques

The Mascot score (M-score) is one of the conventional validity measures in data base identification of peptides and proteins by MS/MS data. Although tremendously useful, M-score has a number of limitations. For the same MS/MS data, M-score may change if the protein data base is expanded. A low M-value may not necessarily mean poor match but rather poor MS/MS quality. In addition M-score does not fully utilize the advantage of combined use of complementary fragmentation techniques collisionally activated dissociation (CAD) and electron capture dissociation (ECD). To address these issues, a new data base-independent scoring method (S-score) was designed that is based on the maximum length of the peptide sequence tag provided by the combined CAD and ECD data. The quality of MS/MS spectra assessed by S-score allows poor data (39% of all MS/MS spectra) to be filtered out before the data base search, speeding up the data analysis and eliminating a major source of false positive identifications. Spectra with below threshold M-scores (poor matches) but high S-scores are validated. Spectra with zero M-score (no data base match) but high S-score are classified as belonging to modified sequences. As an extension of S-score, an extremely reliable sequence tag was developed based on complementary fragments simultaneously appearing in CAD and ECD spectra. Comparison of this tag with the data base-derived sequence gives the most reliable peptide identification validation to date. The combined use of M- and S-scoring provides positive sequence identification from >25% of all MS/MS data, a 40% improvement over traditional M-scoring performed on the same Fourier transform MS instrumentation. The number of proteins reliably identified from Escherichia coli cell lysate hereby increased by 29% compared with the traditional M-score approach. Finally S-scoring provides a quantitative measure of the quality of fragmentation techniques such as the minimum abundance of the precursor ion, the MS/MS of which gives the threshold S-score value of 2.

Determination of Aberrant O-Glycosylation in the IgA1 Hinge Region by Electron Capture Dissociation Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry

In a number of human diseases of chronic inflammatory or autoimmune character, immunoglobulin molecules display aberrant glycosylation patterns of N- or O-linked glycans. In IgA nephropathy, IgA1 molecules with incompletely galactosylated O-linked glycans in the hinge region (HR) are present in mesangial immunodeposits and in circulating immune complexes. It is not known whether the Gal deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. To develop experimental approaches to address this question, the synthetic IgA1 hinge region and hinge region from a naturally Gal-deficient IgA1 myeloma protein have been analyzed by 9.4 tesla Fourier transform-ion cyclotron resonance mass spectrometry. Fourier transform-ion cyclotron resonance mass spectrometry offers two complementary fragmentation techniques for analysis of protein glycosylation by tandem mass spectrometry. Infrared multiphoton dissociation of isolated myeloma IgA1 hinge region peptides confirms the amino acid sequence of the de-glycosylated peptide and positively identifies a series of fragments differing in O-glycosylation. To localize sites of O-glycan attachment, synthetic IgA1 HR glycopeptides and HR from a naturally Gal-deficient polymeric IgA1 myeloma protein were analyzed by electron capture dissociation and activated ion-electron capture dissociation. Multiple sites of O-glycan attachment (including sites of Gal deficiency) in myeloma IgA1 HR glycoforms were identified (in all but one case uniquely). These results represent the first direct identification of multiple sites of O-glycan attachment in IgA1 hinge region by mass spectrometry, thereby enabling future characterization at the molecular level of aberrant glycosylation of IgA1 in diseases such as IgA nephropathy.

Structure-Activity Relationships in Defensin Dimers: A NOVEL LINK BETWEEN β-DEFENSIN TERTIARY STRUCTURE AND ANTIMICROBIAL ACTIVITY

Defensins are cationic antimicrobial peptides that have a characteristic six-cysteine motif and are important components of the innate immune system. We recently described a beta-defensin-related peptide (Defr1) that had potent antimicrobial activity despite having only five cysteines. Here we report a relationship between the structure and activity of Defr1 through a comparative study with its six cysteine-containing analogue (Defr1 Y5C). Against a panel of pathogens, we found that oxidized Defr1 had significantly higher activity than its reduced form and the oxidized and reduced forms of Defr1 Y5C. Furthermore, Defr1 displayed activity against Pseudomonas aeruginosa in the presence of 150 mm NaCl, whereas Defr1 Y5C was inactive. By using nondenaturing gel electrophoresis and Fourier transform ion cyclotron resonance mass spectrometry, we observed Defr1 and Defr1 Y5C dimers. Two complementary fragmentation techniques (collision-induced dissociation and electron capture dissociation) revealed that Defr1 Y5C dimers form by noncovalent, weak association of monomers that contain three intramolecular disulfide bonds. In contrast, Defr1 dimers are resistant to collision-induced dissociation and are only dissociated into monomers by reduction using electron capture. This is indicative of Defr1 dimerization being mediated by an intermolecular disulfide bond. Proteolysis and peptide mass mapping revealed that Defr1 Y5C monomers have beta-defensin disulfide bond connectivity, whereas oxidized Defr1 is a complex mixture of dimeric isoforms with as yet unknown inter- and intramolecular connectivities. Each isoform contains one intermolecular and four intramolecular disulfide bonds, but because we were unable to resolve the isoforms by reverse phase chromatography, we could not assign each isoform with a specific antimicrobial activity. We conclude that the enhanced activity and stability of this mixture of Defr1 dimeric isoforms are due to the presence of an intermolecular disulfide bond. This first description of a covalently cross-linked member of the defensin family provides further evidence that the antimicrobial activity of a defensin is linked to its ability to form stable higher order structures.

Detection and characterization of methionine oxidation in peptides by collision-induced dissociation and electron capture dissociation

Electron capture dissociation (ECD) and collision-induced dissociation (CID), the two complementary fragmentation techniques, are demonstrated to be effective in the detection and localization of the methionine sulfoxide [Met(O)] residues in peptides using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. The presence of Met(O) can be easily recognized in the low-energy CID spectrum showing the characteristic loss of methanesulfenic acid (CH 3SOH, 64 Da) from the side chain of Met(O). The position of Met(O) can then be localized by ECD which is capable of providing extensive peptide backbone fragmentation without detaching the labile Met(O) side chain. We studied CID and ECD of several Met(O)-containing peptides that included the 44-residue human growth hormone-releasing factor (GRF) and the human atrial natriuretic peptide (ANP). The distinction and complementarity of the two fragmentation techniques were particularly remarkable in their effects on ANP, a disulfide bond-containing peptide. While the predominant fragmentation pathway in CID of ANP was the loss of CH 3SOH (64 Da) from the molecular ion, ECD of ANP resulted in many sequence-informative products, including those from cleavages within the disulfide-bonded cyclic structure, to allow for the direct localization of Met(O) without the typical procedures for disulfide bond reduction followed by SH alkylation.